Early genetic diagnosis of clarithromycin resistance in Helicobacter pylori

BACKGROUND The drug resistance rate of clinical Helicobacter pylori(H.pylori)isolates has increased.However,the mechanism of drug resistance remains unclear.In this study,drug-resistant H.pylori strains were isolated from different areas and different populations of Chinese for genomic analysis.AIM To investigate drug-resistant genes in H.pylori and find the genes for the early diagnosis of clarithromycin resistance.METHODS Three drug-resistant H.pylori strains were isolated from patients with gastritis in Bama County,China.Minimal inhibitory concentrations of clarithromycin,metronidazole,and levofloxacin were determined and complete genome sequencing was performed with annotation.Hp1181 and hp1184 genes were found in these strains and then detected by reverse transcription polymerase chain reaction.The relationships between hp1181 or hp1184 and clarithromycin resistance were ascertained with gene mutant and drug-resistant strains.The homology of the strains with hp26695 was assessed through complete genome detection and identification.Differences in genome sequences,gene quantity,and gene characteristics were detected amongst the three strains.Prediction and analysis of the function of drug-resistant genes indicated that the RNA expression of hp1181 and hp1184 increased in the three strains,which was the same in the artificially induced clarithromycin-resistant bacteria.After gene knockout,the drug sensitivity of the strains was assessed.RESULTS The strains showing a high degree of homology with hp26695,hp1181,and hp1184 genes were found in these strains;the expression of the genes hp1184 and hp1181 was associated with clarithromycin resistance.CONCLUSION Hp1181 and hp1184 mutations may be the earliest and most persistent response to clarithromycin resistance,and they may be the potential target genes for the diagnosis,prevention,and treatment of clarithromycin resistance.CONCLUSION Hp1181 and hp1184 mutations may be the earliest and most persistent response to clarithromycin resistance,and they may be the potential target genes for the diagnosis,prevention,and treatment of clarithromycin resistance.

Establishment of a Simple and Useful Way for Preimplantation Genetic Diagnosis of Chromosomal Diseases

In order to establish a simple and useful way for preimplantation genetic diagnosis (PGD) of chromosomal diseases in general IVF laboratory, the methods that are most commonly used in the embryo biopsy, fixation of blastomere and fluorescence in situ hybridization were compared. The three aspects of PGD were analyzed respectively. There was no significant difference in further de- velopment capacity of embryos between mechanical (79.7%) and chemical biopsy group (78.6%) (P>0.05). In this study, more cells were successfully fixed with the Tween/HCL method (93.8%) than with the methanol/acetic acid method (80.5%, P<0.05). There was no significant difference in cyto- plasm remains between methanol/acetic acid method and Tween/HCL method (P>0.05). The hy- bridization efficiency of fluorescence in situ hybridization was 89.5% in successive denaturation method and 90.9% in codenaturation method with the difference being not significant (P>0.05). In conclusion, the mechanical or chemical method, Tween/HCL fixation method and codenaturation fluorescence in situ hybridization method can constitute a simple and useful way for PGD of chro- mosomal diseases.

Atelosteogenesis Type 2/Diastrophic Dysplasia Phenotypic Spectrum: From Prenatal to Preimplantation Genetic Diagnosis

Atelosteogenesis type II (AO2) and diastrophic dysplasia (DTD) are two recessively inherited, severe skeletal dysplasias caused by mutations in the SLC26A2 gene. AO2 is an invariably lethal condition, while DTD patients may reach adult life, although both diseases have overlapping diagnostic features. Here we report a patient with an intermediate phenotype between AO2 and DTD and present the successful application of preimplantation genetic diagnosis (PGD) in this situation. Sequencing of SLC26A2 alleles in the infant identified two compound heterozygous mutations, p.Arg178Ter and p.Arg279Trp, of paternal and maternal origin, respectively. At request from the parents, PGD was developed by haplotype mapping of parental SLC26A2 alleles in eleven five-day embryos. Transference to the mother was attempted twice, finally resulting in pregnancy and delivery of a healthy baby. This exemplifies the utility of PGD for inherited lethal conditions with a significant risk of recurrence, and highlights the importance of accurate diagnosis of skeletal dysplasias with prenatal manifestation.

The Performance of Whole Genome Amplification Methods and Next-Generation Sequencing for Pre-Implantation Genetic Diagnosis of Chromosomal Abnormalities

Reliable and accurate pre-implantation genetic diagnosis （PGD） of patient＇s embryos by next-generation sequencing （NGS） is dependent on efficient whole genome amplification （WGA） of a representative biopsy sample. However, the performance of the current state of the art WGA methods has not been evaluated for sequencing. Using low template DNA （15 pg） and single cells, we showed that the two PCR-based WGA systems SurePlex and MALBAC are superior to the REPLI-g WGA multiple displacement amplification （MDA） system in terms of consistent and reproducible genome coverage and sequence bias across the 24 chromosomes, allowing better normalization of test to reference sequencing data. When copy number variation sequencing （CNV-Seq） was applied to single cell WGA products derived by either SurePlex or MALBAC amplification, we showed that known disease CNVs in the range of 3-15 Mb could be reliably and accurately detected at the correct genomic positions. These findings indicate that our CNV-Seq pipeline incorporating either SurePlex or MALBAC as the key initial WGA step is a powerful methodology for clinical PGD to identify euploid embryos in a patient＇s cohort for uterine transplantation,

Clinical applications of MARSALA for preimplantation genetic diagnosis of spinal muscular atrophy

Conventional PCR methods combined with linkage analysis based on short tandem repeats （STRs） or Karyomapping with single nucleotide polymorphism （SNP） arrays, have been applied to preimplantation genetic diagnosis （PGD） for spinal muscular atrophy （SMA）, an autosome recessive disorder. However, it has limitations in SMA diagnosis by Karyomapping, and these methods are unable to distinguish wild- type embryos with carriers effectively. Mutated allele revealed by sequencing with aneuploidy and linkage analyses （MARSALA） is a new method allowing embryo selection by a one-step next-generation sequencing （NGS） procedure, which has been applied in PGD for both autosome dominant and X-linked diseases in our group previously. In this study, we carried out PGD based on MARSALA for two carrier families with SMA affected children. As a result, one of the couples has given birth to a healthy baby free of mutations in SMA-causing gene. It is the first time that MARSALA was applied to PGD for SMA, and we can distinguish the embryos with heterozygous deletion （carriers） from the wild-type （normal） ones accurately through this NGS-based method. In addition, direct mutation detection allows us to identify the affected embryos （homozygous deletion）, which can be regarded as probands for linkage analysis, in case that the affected family member is absent, In the future, the NGS-based MARSALA method is expected to be used in PGD for all monogenetic disorders with known pathogenic gene mutation.

Study on Genetic Diagnosis of Spinal Muscular Atrophy

An improved method of mismatching polymerase chain reactionrestrictive fragment length polymorphisms(PCR-RFLP) was performed in our lab for genetic diagnosis of spinal muscular atrophy(SMA). PCR amplification and restriction endonuclease digestion of exons 7 and 8 permit distinction of the telomeric survival motor neuron (SMN) gene and its centromeric copy. Lack of a PCR product from either exon and from either gene is indicative of homozygous deletion of that sequence, and a high correlation with clinical SMA. Our data showed: 9 cases in 10 presumed SMA children were positive, i.e. deletion of telomeric SMN gene. One case was negative. 20 cases of normal familial members and 20 cases of normal health persons were all negative. Our results matched the criteria and reports of foreign countries. The method we used is highly specific, sensitive and reliable and is suitable for genetic diagnosis of SMA and its prenatal diagnosis.

A New Next-Generation Sequencing-Based Assay for Concurrent Preimplantation Genetic Diagnosis of Charcot-Marie-Tooth Disease Type 1A and Aneuploidy Screening

Charcot-Marie-Tooth （CMT） disease is the most common hereditary neuropathy, with a population prevalence of 1 in 2500. CMT disease type 1A （CMT1A）, accounting for ~70% of CMT1 cases and ~ 50% of all CMT cases, is transmitted in an autosomal dominant manner. CMT1A maps to chromo- some 17pl 1.2 and is caused, in the majority of cases, by a 1.4- Mb tandem duplication that includes the peripheral myelin protein22 （PMP22） gene （Li et al., 2013）. The disease usually presents in the first 20 years of age, causing difficulty in walking or running, distal symmetrical muscle weakness and wasting, and sensory loss （van Paassen et al., 2014）.

Genetic diagnosis for heavy typhoon rainfall attenuated by Fujian landfall

This study used the dynamic synthetic analysis method to analyze the causes of attenuated heavy rainfall from a westward moving typhoon after landfall over Fujian by focusing on the genetic diagrgnosis of the strongest 12 h rainstorms based on typhoon data obtained from the Shanghai Typhoon Institute,precipitation data from Fujian Province,and NCEP reanalysis data from the United States.The results showed that:(1)the environmental field of the westward moving typhoon benefits the long-term maintenance of convergence in coastal areas,which provides synoptic scale forcing for rainstorm intensification along the southeastern coast;(2)the southwest jet in the boundary layer transports warm water vapor from low latitudes into the eastern circulation of typhoon;the water vapor peak occurs 6 h before the strongest rainstorm and can be used as a reference index to predict heavy rainstorms;(3)the high altitude strong divergence center is located at 100-150 hPa,and the strong convergence center is located near 925-950 hPa in the boundary layer,which is higher(lower)than the 200 hPa divergence layer(850 hPa convergence layer)commonly used in professional work;(4)warm and wet advection in the boundary layer transports unstable energy and weak cold air southward,strengthens the baroclinic pressure,increases the latent heat flux on the sea surface,and plays a significant role in triggering and developing mesoscale convective clouds along the southeast coast.

Preliminary Investigation on the Role of Vitrification in Pre-Implantation Genetic Diagnosis

Objective To investigate the feasibility of vitrification of blastocysts following blastomere biopsy. Methods Among patients undergoing pre-implantation genetic diagnosis （PGD）, artificial shrinkage of the blastocoelic cavity and subsequent vitrification of applicable surplus blastocysts after day-3 blastomere biopsy were performed. According to patient requirements, thawed blastocysts were transferred into patients due to pregnancy failure after fresh embryo transfer, ectopic pregnancy, ovarian hyperstimulation. Results Twenty-four PGD cycles were carried out. According to genetic diagnosis and the development of blastocysts, transfer was cancelled in 7 cycles due to absence of applicable embryos or ovarian hyperstimulation. In the remaining 17 cycles, 26 blastocysts were thawed and transferred, which resulted in 13 implanted （50.0%）. Clinical pregnancies were observed in 11 patients （64.71%）. Following transfer, 30 applicable blastocysts in 10 cycles were cryopreserved. Six patients received transfer of thawed blastocysts. All 8 thawed embryos survived and were transferred, and singleton pregnancies occurred in 5 patients. Two women delivered healthy infants and 3 pregnancies are ongoing. Conclusion Vitrification with artificial shrinkage is effective for preserving blastocysts following blastomere biopsy.

Improving genetic diagnosis of Mendelian disease with RNA sequencing:a narrative review

Targeted sequencing and whole exome sequencing are the most common approaches used to detect causative variants in Mendelian diseases;however, using DNA-based sequencing techniques, the current molecular diagnostic yield is at best 50%. In recent years, RNA sequencing has been shown to be able to provide a genetic diagnosis in patients whose conditions were previously unable to be identified by DNA analysis. RNA sequencing can reveal expression outliers, aberrant splicing events, allele-specific expression, and new pathogenic variants, and as such can complement and expand on the traditional genomic methods used to diagnose Mendelian diseases. Therefore, RNA sequencing is expected to become a routine method for genetic diagnosis in the future. This article reviews the applications and challenges of RNA sequencing in the genetic diagnosis of Mendelian diseases.

Preimplantation genetic diagnosis of hereditary hearing loss:a narrative review

Preimplantation genetic diagnosis(PGD)uses molecular biological techniques to genetically diagnose embryos beforein vitro fertilization.The information obtained through PGD can help clinicians select healthy embryos for implantation,prevent the transmission of inherited diseases and help affected families have healthy children.This paper reviews the development of PGD technology,the history of its application to hereditary hearing loss,and the general process of how PGD is applied to screen for hereditary hearing loss.The aim of this review is to demonstrate the reliability of PGD in the primary prevention of hereditary hearing loss,assist clinicians in counseling patients at risk of transmitting an inherited disease,and explore the journey from PGD toin vitro fertilization.Given that the application of PGD technology to hereditary hearing loss varies in different countries and regions,there is still a long way to go before PGD is routinely applied for the primary prevention of hereditary hearing loss.

preimplantation genetics diagnosis译为“植入前遗传诊断”为妥

1.implantation按1993年《全国自然科学名词审定委员会公布、胚胎学名》第126页,审定“implantation”译为“植入”(胚泡进入子宫内膜的过程)编号为02.078。 2.人民卫生出版社出版的《英汉医学词汇》第706页和《汉英医学词汇》均将implantation译为“植入”(胚泡在子宫内)。

Genetic analysis of Chinese families reveals a novel truncation allele of the retinitis pigmentosa GTPase regulator gene

AIMTo make comprehensive molecular diagnosis for retinitis pigmentosa (RP) patients in a consanguineous Han Chinese family using next generation sequencing based Capture-NGS screen technology.

Gene diagnosis of infantile neurofibromatosis type I:A case report

BACKGROUND Neurofibromatosis is an autosomal dominant genetic disorder with various manifestations.Systemic multiple neurofibromatosis is rare in infancy.The disease is difficult to identify in the early stage,and it is prone to misdiagnosis and missed diagnosis.In the presence of lower limb swelling with subcutaneous nodules of unknown cause,café-au-lait spots,and axillary freckles,this disease must be considered.This report presents the clinical manifestations,early detection,diagnosis and treatment,and prognosis of infantile neurofibromatosis type I(NF1).CASE SUMMARY The clinical manifestations,imaging examinations,and gene results of a 3-mo-old male infant with NF1 were analyzed retrospectively.He had“swelling of both legs”at the onset and developed café-au-lait spots,axillary freckles,and multiple neurofibromas later.He had a family history of similar conditions.Gene detection showed a heterozygous mutation of c.4537C>T in the NF1 gene,leading to a nonsense mutation of amino acids(p.R1513x),which originated from the mother of the infant.He was diagnosed with NF1.CONCLUSION Gene diagnosis plays an important role in the early diagnosis of NF1.

染色体异常与男性不育

Infertility in humans is surprisingly common occurring in approximately 15% of the population wishing to start a family. Despite this, the molecular and genetic factors underlying the cause of infertility remain largely undiscovered. Nevertheless, more and more genetic factors associated with infertility are being identified. This review will focus on our current understanding of the chromosomal basis of male infertility specifically： chromosomal aneuploidy, structural and numerical karyotype abnormalities and Y chromosomal microdeletions. Chromosomal aneuploidy is the leading cause of pregnancy loss and developmental disabilities in humans. Aneuploidy is predominantly maternal in origin, but concerns have been raised regarding the safety of intracytoplasmic sperm injection as infertile men have significantly higher levels of sperm aneuploidy compared to their fertile counterparts. Males with numerical or structural karyotype abnormalities are also at an increased risk of producing aneuploid sperm. Our current understanding of how sperm aneuploidy translates to embryo aneuploidy will be reviewed, as well as the application of preimplantation genetic diagnosis （PGD） in such cases. Clinical recommendations where possible will be made, as well as discussion of the use of emerging array technology in PGD and its potential applications in male infertility.

Single Cell Analysis of Dystrophin and SRY Gene by Using Whole Genome Amplification

ve To develop a reliable and sensitive method for detection of sex and multi-loci of Duchenne muscular dystrophy (DMD) gene in single cell

Examination of Huntington's disease in a Chinese family

We report brain imaging and genetic diagnosis in a family from Wuhan, China, with a history of Huntington＇s disease. Among 17 family members across three generations, four patients （Ⅱ2, Ⅱ6, Ⅲ5, and Ⅲ9） show typical Huntington＇s disease, involuntary dance-like movements. Magnetic resonance imaging found lateral ventricular atrophy in three members （Ⅱ2, Ⅱ6, and Ⅲ5）. Moreover, genetic analysis identified abnormally amplified CAG sequence repeats （〉 40） in two members （Ⅲ5 and Ⅲ9）. Among borderline cases, with clinical symptoms and brain imaging features of Huntington＇s disease, two cases were identified （Ⅱ2 and Ⅱ6）, but shown by mutation analysis for CAG expansions in the important transcript 15 gene, to be non-Huntington＇s disease. Our findings suggest that clinical diagnosis of Huntington＇s disease requires a combination of clinical symptoms, radiological changes, and genetic diagnosis.

The forty years of medical genetics in China

Medical genetics is the newest cutting-edge discipline that focuses on solving medical problems using genetics knowledge and methods. In China, medical genetics research activities initiated from a poor inner basis but a prosperous outer environment. During the 40 years of reform and opening-up policy,Chinese scientists contributed significantly in the field of medical genetics, garnering considerable attention worldwide. In this review, we highlight the significant findings and/or results discovered by Chinese scientists in monogenic diseases, complex diseases, cancer, genetic diagnosis, as well as gene manipulation and gene therapy. Due to these achievements, China is widely recognized to be at the forefront of medical genetics research and development. However, the significant progress and development that has been achieved could not have been accomplished without sufficient funding and a wellconstructed logistics network. The successful implementation of translational and precise medicine sourced from medical genetics will depend on an open ethics policy and intellectual property protection,along with strong support at the national industry level.

Outcome of Couples with Reciprocal Translocation Carrier Undergoing the First Preimplantation Genetic Testing Cycles

Background: Reciprocal translocation(RCP) causes male infertility and female recurrent pregnancy loss. Male and female carriers have different responses to meiotic disturbances. Gender difference in outcomes of the RCP couples undergoing preimplantation genetic testing(PGT) is unknown.Methods: We conducted a retrospective analysis of 238 RCP couples(124 female and 114 male carriers) divided by gender of carrier from March 2014 to March 2017. Blastocysts were divided by day 5 and day 6. Females were divided into older(≥38 years) and younger(<38 years). Logistic regression was fitted for the relationship between gender of carriers and euploidy. Euploidy rate of each group, pregnancy rate, and live birth rate between different genders were analyzed.Results: The sperm live rate, forward motile sperm rate, and normal morphology rate of serum in male RCP group were significantly decreased. The euploidy rate was 30.30% in female group and 34.90% in male group(P = 0.131); 34.50% in day 5 group and 27.50% in day 6 group(P = 0.039); 33.40% in age <38 years group and 22.40% in age ≥38 years group(P = 0.063). Day 5(odds ratio [OR] = 1.388, 95% confidence interval [CI ] = 1.012–1.904; P = 0.042) and younger age(OR = 1.753, 95% CI = 0.97–3.17; P = 0.063) were associated with euploidy. The clinical pregnancy rate(37.90% vs. 41.20%), ongoing pregnancy rate(33.10% vs. 37.70%), and live birth rate(25.80% vs. 31.60%) per initiated were not significantly different in two gender groups.Conclusions: Although gender influence is not significant, couples with male carrier showed better clinical outcomes. The embryo growing rate and female age are important predictions estimating euploidy in RCP couples.

Beyond monogenetic rare variants:tackling the low rate of genetic diagnoses in predominantly antibody deficiency

Predominantly antibody deficiency(PAD)is the most prevalent form of primary immunodeficiency,and is characterized by broad clinical,immunological and genetic heterogeneity.Utilizing the current gold standard of whole exome sequencing for diagnosis,pathogenic gene variants are only identified in less than 20% of patients.While elucidation of the causal genes underlying PAD has provided many insights into the cellular and molecular mechanisms underpinning disease pathogenesis,many other genes may remain as yet undefined to enable definitive diagnosis,prognostic monitoring and targeted therapy of patients.Considering that many patients display a relatively late onset of disease presentation in their 2^(nd) or 3^(rd) decade of life,it is questionable whether a single genetic lesion underlies disease in all patients.Potentially,combined effects of other gene variants and/or non-genetic factors,including specific infections can drive disease presentation.In this review,we define(1)the clinical and immunological variability of PAD,(2)consider how genetic defects identified in PAD have given insight into B-cell immunobiology,(3)address recent technological advances in genomics and the challenges associated with identifying causal variants,and(4)discuss how functional validation of variants of unknown significance could potentially be translated into increased diagnostic rates,improved prognostic monitoring and personalized medicine for PAD patients.A multidisciplinary approach will be the key to curtailing the early mortality and high morbidity rates in this immune disorder.